The Co-mutational Spectrum Determines the Therapeutic Response in Murine FGFR2 Fusion-Driven Cholangiocarcinoma.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer and a highly lethal malignancy. Chemotherapeutic options are limited, but a considerable subset of patients harbors genetic lesions for which targeted agents exist. Fibroblast growth factor receptor 2 (FGFR2) fusions belong to the most frequent and therapeutically relevant alterations in ICC, and the first FGFR inhibitor was recently approved for the treatment of patients with progressed, fusion-positive ICC. Response rates of up to 35% indicate that FGFR-targeted therapies are beneficial in many but not all patients. Thus far, no established biomarkers exist that predict resistance or response to FGFR-targeted therapies in patients with ICC. APPROACH AND RESULTS: In this study, we use an autochthonous murine model of ICC to demonstrate that FGFR2 fusions are potent drivers of malignant transformation. Furthermore, we provide preclinical evidence that the co-mutational spectrum acts not only as an accelerator of tumor development, but also modifies the response to targeted FGFR inhibitors. Using pharmacologic approaches and RNA-interference technology, we delineate that Kirsten rat sarcoma oncogene (KRAS)-activated mitogen-activated protein kinase signaling causes primary resistance to FGFR inhibitors in FGFR2 fusion-positive ICC. The translational relevance is supported by the observation that a subset of human FGFR2 fusion patients exhibits transcriptome profiles reminiscent of KRAS mutant ICC. Moreover, we demonstrate that combination therapy has the potential to overcome primary resistance and to sensitize tumors to FGFR inhibition. CONCLUSIONS: Our work highlights the importance of the co-mutational spectrum as a significant modifier of response in tumors that harbor potent oncogenic drivers. A better understanding of the genetic underpinnings of resistance will be pivotal to improve biomarker-guided patient selection and to design clinically relevant combination strategies.

Risk estimation for biliary tract cancer: Development and validation of a prognostic score.

BACKGROUND & AIMS: Biliary tract cancer is a rare tumour entity characterized by a poor prognosis. We aimed to identify prognostic factors and create a prognostic score to estimate survival. METHODS: Clinical data of the training set, consisting of 569 patients treated from 2000 to 2010 at Hannover Medical School, were analysed. A prognostic model defining three prognostic risk groups was derived from Cox regression analyses. The score was applied and validated in an independent cohort of 557 patients from four different German centres. RESULTS: Median overall survival (OS) was 14.5 months. If complete resection was performed, the patients had a significantly improved OS (23.9 months; n=242) as compared to patients with non-resectable tumours (9.1 months; n=329, P<.0001). Based on univariable and multivariable analyses of clinical data, a prognostic model was created using variables available before treatment. Those were age, metastasis, C-reactive protein (CRP), international normalized ratio (INR) and bilirubin. The prognostic score distinguished three groups with a median OS of 21.8, 8.6 and 2.6 months respectively. The validation cohort had a median OS of 20.2, 14.0 and 6.5 months respectively. The prognostic impact of the score was independent of the tumour site and of treatment procedures. CONCLUSIONS: Here, we identified prognostic factors and propose a prognostic score to estimate survival, which can be applied to all patients independent of tumour site and before initial treatment. Further validation in prospective trials is required.

Patterns and challenges of treatment sequencing in patients with hepatocellular carcinoma: Experience from a German referral center.

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers. Several local and systemic therapies are available for patients with HCC depending on the stage of the disease. In clinical practice, treatment decision-making, and sequencing may be very heterogeneous. METHODS: In this study, we retrospectively analyzed treatment algorithms in 2101 patients with HCC treated from 2000 to 2015 at Hannover Medical School, Germany. RESULTS: Transarterial chemoembolization was the most common initial treatment (n = 545; 25.9%), followed by resection (n = 435, 20.7%), local-ablative procedures (n = 283, 13.5%), systemic therapies (n = 275, 13.1%), and liver transplantation (n = 52; 2.5%). Most patients were treated only once (n = 960; 59.6%). A total of 433 (26.9%) and 160 (9.9%) patients received a second line and third line treatment after recurrent or progressive disease. Patients with more than one treatment line were diagnosed at significantly earlier disease stages (P < 0.001). Using binary logistic regression, AFP ≤ 200 µg/L, albumin > 36 g/L, and small tumor size (≤50 mm) were identified as predictors of achieving more than one treatment line. Subsequent treatment stage migration to a therapy suggested for the next advanced stage occurred only in 56.9%, whereas 43.1% received treatments suggested for earlier disease stages. Only 16% of all treated patients received systemic therapy in the salvage setting. CONCLUSION: Most patients were treated only once, and only a minority of patients received systemic treatment. The high dropout rate for subsequent therapies needs to be considered within therapy decision-making. There is an urgent need for prospective studies to define the best time point when to switch patients from local to systemic therapies.

Microenvironment-induced restoration of cohesive growth associated with focal activation of P-cadherin expression in lobular breast carcinoma metastatic to the colon.

Invasive lobular carcinoma (ILC) is a special breast cancer type characterized by noncohesive growth and E-cadherin loss. Focal activation of P-cadherin expression in tumor cells that are deficient for E-cadherin occurs in a subset of ILCs. Switching from an E-cadherin deficient to P-cadherin proficient status (EPS) partially restores cell-cell adhesion leading to the formation of cohesive tubular elements. It is unknown what conditions control EPS. Here, we report on EPS in ILC metastases in the large bowel. We reviewed endoscopic colon biopsies and colectomy specimens from a 52-year-old female (index patient) and of 18 additional patients (reference series) diagnosed with metastatic ILC in the colon. EPS was assessed by immunohistochemistry for E-cadherin and P-cadherin. CDH1/E-cadherin mutations were determined by next-generation sequencing. The index patient's colectomy showed transmural metastatic ILC harboring a CDH1/E-cadherin p.Q610* mutation. ILC cells displayed different growth patterns in different anatomic layers of the colon wall. In the tunica muscularis propria and the tela submucosa, ILC cells featured noncohesive growth and were E-cadherin-negative and P-cadherin-negative. However, ILC cells invading the mucosa formed cohesive tubular elements in the intercryptal stroma of the lamina propria mucosae. Inter-cryptal ILC cells switched to a P-cadherin-positive phenotype in this microenvironmental niche. In the reference series, colon mucosa infiltration was evident in 13 of 18 patients, one of which showed intercryptal EPS and conversion to cohesive growth as described in the index patient. The large bowel is a common metastatic site in ILC. In endoscopic colon biopsies, the typical noncohesive growth of ILC may be concealed by microenvironment-induced EPS and conversion to cohesive growth.

Concordance in detection of microsatellite instability by PCR and NGS in routinely processed tumor specimens of several cancer types.

BACKGROUND: Microsatellite instability (MSI) occurs in several cancer types and is commonly used for prognosis and as a predictive biomarker for immune checkpoint therapy. METHODS: We analyzed n = 263 formalin-fixed paraffin-embedded (FFPE) tumor specimens (127 colorectal cancer (CRC), 55 endometrial cancer (EC), 33 stomach adenocarcinoma (STAD), and 48 solid tumor specimens of other tumor types) with a capillary electrophoresis based multiplex monomorphic marker MSI-PCR panel and an amplicon-based NGS assay for microsatellite instability (MSI+). In total, n = 103 (39.2%) cases with a known defect of the DNA mismatch repair system (dMMR), determined by a loss in protein expression of MSH2/MSH6 (n = 48, 46.6%) or MLH1/PMS2 (n = 55, 53.4%), were selected. Cases with an isolated loss of MSH6 or PMS2 were excluded. RESULTS: The overall sensitivity and specificity of the NGS assay in comparison with the MSI-PCR were 92.2% and 98.8%. With CRC cases a nearly optimal concordance was reached (sensitivity 98.1% and specificity 100.0%). Whereas EC cases only show a sensitivity of 88.6% and a specificity of 95.2%, caused by several cases with instability in less than five monomorphic markers, which could be difficult to analyze by NGS (subtle MSI+ phenotype). CONCLUSIONS: MSI analysis of FFPE DNA by NGS is feasible and the results show a high concordance in comparison with the monomorphic marker MSI-PCR. However, cases with a subtle MSI+ phenotype, most frequently manifest in EC, have a risk of a false-negative result by NGS and should be preferentially analyzed by capillary electrophoresis.

Molecular diagnostics and therapies for gastrointestinal tumors: a real-world experience.

PURPOSE: Several targeted agents demonstrated efficacy in early clinical trials for gastrointestinal (GI) cancers, but in many cases, phase-III trials and/or approval by the European Medicines Agency (EMA) are lacking. The primary focus of this study was to assess the regulatory processes associated with use and reimbursement of off-label treatment in precision oncology and to evaluate the benefit of targeted therapy in a real-world population in Germany. METHODS: Our cohort comprises 137 patients with GI cancers and is biased towards cancer entities with a high frequency of known targetable alterations, such as cholangiocarcinoma. Genetic testing was used to identify molecular targets, and therapy response was evaluated based on CT scans. RESULTS: A molecular target for precision oncology was identified in 53 patients and 43 requests for cost coverage were submitted to health insurance companies. 60% of the requests received approval after initial application and another 7% after appeal. Half of the rejected requests were denied despite ESCAT IA level evidence. The median time between initiation of molecular testing and start of therapy was 75 days. 35 patients received matched targeted therapies (n = 28) or, in the case of MSI, immunotherapy (IO) (n = 7). We observed a trend in favor of molecular therapy when compared to the immediate prior treatment. CONCLUSION: Relevant treatment options were identified by molecular testing in a significant subset of patients. When targeted therapies that lack EMA approval are considered, treatment initiation may be delayed by the duration of the molecular analysis and the regulatory processes.

Inter-observer agreement for the histological diagnosis of invasive lobular breast carcinoma.

Invasive lobular breast carcinoma (ILC) is the second most common breast carcinoma (BC) subtype and is mainly driven by loss of E-cadherin expression. Correct classification of BC as ILC is important for patient treatment. This study assessed the degree of agreement among pathologists for the diagnosis of ILC. Two sets of hormone receptor (HR)-positive/HER2-negative BCs were independently reviewed by participating pathologists. In set A (61 cases), participants were provided with hematoxylin/eosin (HE)-stained sections. In set B (62 cases), participants were provided with HE-stained sections and E-cadherin immunohistochemistry (IHC). Tumor characteristics were balanced. Participants classified specimens as non-lobular BC versus mixed BC versus ILC. Pairwise inter-observer agreement and agreement with a pre-defined reference diagnosis were determined with Cohen's kappa statistics. Subtype calls were correlated with molecular features, including CDH1/E-cadherin mutation status. Thirty-five pathologists completed both sets, providing 4,305 subtype calls. Pairwise inter-observer agreement was moderate in set A (median κ = 0.58, interquartile range [IQR]: 0.48-0.66) and substantial in set B (median κ = 0.75, IQR: 0.56-0.86, p < 0.001). Agreement with the reference diagnosis was substantial in set A (median κ = 0.67, IQR: 0.57-0.75) and almost perfect in set B (median κ = 0.86, IQR: 0.73-0.93, p < 0.001). The median frequency of CDH1/E-cadherin mutations in specimens classified as ILC was 65% in set A (IQR: 56-72%) and 73% in set B (IQR: 65-75%, p < 0.001). Cases with variable subtype calls included E-cadherin-positive ILCs harboring CDH1 missense mutations, and E-cadherin-negative ILCs with tubular elements and focal P-cadherin expression. ILCs with trabecular growth pattern were often misclassified as non-lobular BC in set A but not in set B. In conclusion, subtyping of BC as ILC achieves almost perfect agreement with a pre-defined reference standard, if assessment is supported by E-cadherin IHC. CDH1 missense mutations associated with preserved E-cadherin protein expression, E- to P-cadherin switching in ILC with tubular elements, and trabecular ILC were identified as potential sources of discordant classification.

A novel germline POLE mutation causes an early onset cancer prone syndrome mimicking constitutional mismatch repair deficiency.

In a 14-year-old boy with polyposis and rectosigmoid carcinoma, we identified a novel POLE germline mutation, p.(Val411Leu), previously found as recurrent somatic mutation in 'ultramutated' sporadic cancers. This is the youngest reported cancer patient with polymerase proofreading-associated polyposis indicating that POLE mutation p.(Val411Leu) may confer a more severe phenotype than previously reported POLE and POLD1 germline mutations. The patient had multiple café-au-lait macules and a pilomatricoma mimicking the clinical phenotype of constitutional mismatch repair deficiency. We hypothesize that these skin features may be common to different types of constitutional DNA repair defects associated with polyposis and early-onset cancer.

The effect of adjuvant chemotherapy in patients with intrahepatic cholangiocarcinoma: a matched pair analysis.

PURPOSE: The purpose of this study was to identify prognostic factors of patients with intrahepatic cholangiocarcinoma (ICC) treated with resection and to investigate the effect of adjuvant chemotherapy (CT). METHODS: Patients with ICC diagnosed between 2000 and 2015 treated at Hannover Medical School were included. Clinicopathologic characteristics were analyzed in univariate and multivariate analysis. In a matched pair survival analysis, patients with or without adjuvant CT were matched by these prognostic factors. RESULTS: Two hundred and ten patients were included. Median survival was 28.7 months, 1-, 3-, and 5-year survival rates were 72.8%, 29.6%, and 14.1%, respectively. In multivariate analysis, lymph node involvement (p = 0.006, HR 1.84), larger tumor size (p = 0.013, HR 1.79), vascular invasion (p = 0.038, HR 1.70), and prolongation of prothrombin time (p < 0.001, HR 4.20) were significantly related to poor survival. Thirty-nine patients received adjuvant CT of which 60% had lymph node involvement. Each 25 patients with and without adjuvant CT were matched to the identified prognostic factors. The median survival of patients with adjuvant CT was 33.5 months, compared to 18 months in the control group (p = 0.002). The 1-, 3-, and 5-year survival rates were 96, 36, and 12%, compared to 60, 4, and 0% in non-treated patients. CONCLUSIONS: We identified several prognostic factors for patients with ICC treated with resection. Our data support the use of adjuvant CT in patients with ICC. The results of prospective randomized controlled studies will clarify the role of adjuvant CT in the future.